Derivatives of 3,6-bis-(2-(1-azabicyclo-(3,1,0)hexane))-2,5-piperazinedione

ABSTRACT

1. A COMPOUND OF THE STRUCTURE   1-((R3)1-),2,5-DI(O=),3,6-BIS(1-AZABICYCLO(3,1,0)-   HEX-2-YL-),3-R4,4-R3,6-((R4)1-)-PIPERAZINE   WHEREIN R3 AND R31 ARE EACH HYDROGEN, LOWER ALKYL OF 1 TO 6 CARBON ATOMS, LOWER ALKANOYL OF 2 TO 4 CARBON ATOMS, DILOWER ALKYLAMINOMETHYL WHEREIN THE LOWER ALKYLS CONTAIN 2 TO 5 CARBON ATOMS; R4 AND R41 ARE EACH HYDROGEN OR MERCAPTO.

United States Patent 3 840 542 DERIVATIVES 0F 3, 6 BIS-{2-[1-AZABICYCLO-(3,1,0)HEXANE]}-2,5-PIPERAZINEDIONE Tsung-Ying Shen, Westfield, NormanP. Jensen, Watchnng,

and Arthur F. Wagner, Princeton, N.J., assignors to Merck & Co., Inc.,Rahway, NJ.

No Drawing. Filed Nov. 24, 1971, Ser. No. 201,956

Int. Cl. C07d 51/70 U.S. Cl. 260-268 DK 2 Claims ABSTRACT OF THEDISCLOSURE Novel derivatives of 3,6bis-(Z-piperidinyl)-2,5-piperazinedione and processes for preparing thesame. Specifically the invention is directed to derivatives of3,6-bis-{2- [l-azabicyclo(3,1,0)hexane]}-2,5 piperazinedione. The newcompounds exhibit activity in inhibiting the growth of tumors in mammalsand birds, and in inhibiting the growth of certain microorganisms.

Field of the Invention This invention relates to novel piperazinedionecompounds. More particularly, this invention relates to a new class ofsubstituted 3,6-bis(2-piperidinyl)-2,5-piperazinedione compounds, theacid salts thereof and processes for preparing the same.

Summary of the Invention The novel compounds of this invention can berepresented by the following structural formula:

wherein R and/or R represent hydrogen, loweralkyl, for example, methyl,ethyl, propyl, isopropyl, butyl, tbutyl, hexyl; substituted loweralkyl,for example, haloloweralkyl, such as 2-chloroethy1, 2-bromoethyl,dichloroethyl; loweralkylcarbonyloxyalkyl, for example acetoxymethyl,acetoxyethyl, propionoxymethyl, pivaloyloxymethyl; aroyloxyalkyl,benzoyloxymethyl, p-toloyloxy methyl; hydroxyloweralkyl, for example,2-hydroxyethyl, 2-hydroxypropyl; aralkyl, for example, mononucleararalkyl, benzyl, phenethyl, phenylpropyl, p-fiuorobenzyl,p-chlorobenzyl, p-methoxybenzyl, p-nitrobenzyl, o-methoxybenzyl,3,4,5-trimethoxybenzyl; acyl, for example loweralkanoyl, such as acetyl,propionyl, butyryl; haloloweralkanoyl, such as chloroacetyl,trichloroacetyl, trifluoroacetyl, chloropropionyl; cycloloweralkanoyl,such as cyclopropylcarbonyl, cyclohexylcarbonyl; aroyl, such as benzoyl,p-chlorobenzoyl, toluoyl, p-fluorobenzoyl, p-nitrobenzoyl,p-phenoxybenzoyl, xyloyl; loweralkyloxycarbonyl, for example,ethoxycarbonyl, propoxycarbonyl, tbutyloxycarbonyl;haloalkyloxycarbonyl, for example 6, 8,

3,840,542 Patented Oct. 8, 1974 at the 4,5 or 5,6 positions; R and/or Rrepresent hydrogen, loweralkyl, for example methyl, ethyl, propyl,isopropyl, butyl, hexyl; loweralkanoyl, for example acetyl, propionyl,butyryl, trifluoroacetyl, diloweralkylaminomethyl, e.g.dimethylaminomethyl, diethylaminomethyl, dipropylaminomethyl,dibutylaminomethyl, methylethylaminomethyl, methylbutylaminomethyl,cycloaminomethyl, e.g. morpholinomethyl, piperidinomethyl, nitroso; Rand/or R represents hydrogen, mercapto, epidithio (SS); R R and R ',Rjoined together through -CH acid addition salts thereof whereinloweralkyl represents from 1 to 6 carbon atoms and with the proviso thatwhen R and R are chloro, R R R R R and R are not hydrogen.

The compounds of this invention can be prepared from 3,6-bis-(5chloro-2-piperidinyl) 2,5 piperazinedione which can be prepared byfermentation. The starting material may be produced by either surface orsubmerged cultures, however, it is presently preferred to carry out thefermentation in the submerged state. Small scale fermentation batchesmay be conveniently prepared by placing suitable quantities of nutrientmedium in flasks, sterilizing the fiasks and contents by heating toabout C. for twenty minutes, inoculating the flasks with vegetativecultures of a 3,6-bis-(5 chloro-Z-piperidinyl)-2,5-piperazinedioneproducing strain of Streptomyces griseoluteus, loosely sealing theflasks with cotton and allowing fermentation to proceed on a shaker in aconstant room at 28 C. for 3-5 days. Larger fermentation batches may beprepared, using suitably sized tanks provided with an agitator and ameans of aerating the fermentation medium. In this method the medium andtanks containing the sterilized medium is inoculated with a vegetativeculture. The fermentation is allowed to proceed from 2-4 days withconstant agitation or aeration of the nutrient medium at a constanttemperature of about 28 C. In carrying out the fermentation according tothis process it may be desirable to add a small amount of a suitableantifoaming agent. Suitable agents may include soybean oil, castor oil,1% octadecanol in mineral oil, or a polymerized propylene glycol such aspolyglycol 2,000. These agents will thus control any excess foaming thatmay occur in the fermentation broth during fermentation.

Aqueous media, such as those employed for the production of antibiotics,are suitable for producing 3,6-bis-(5- chloro-2-piperidinyl) 2,5piperazinedione. Such media contain sources of carbon and nitrogenassimilable by the microorganism and inorganic salts. In addition, the

fermentation media contain traces of metals necessary for the growth ofthe microorganism which are commonly supplied as impurities incidentalto the addition of other constituents of the medium.

In general, carbohydrates such as sugars, for example, glucose, maltose,fructose, and the like, and starches such as grains, for example oatsand rye, corn starch, corn meal, and the like, can be used either aloneor in combination as sources of assimilable carbon in the nutrientmedium. The exact quantity of the carbohydrate source or sourcesutilized in the medium will depend in part upon the other ingredients ofthe medium, but it is usually found that an amount of carbohydratebetween about 1 and 6% by weight of the medium is satisfactory. Thesecarbon sources can be used individually or several such carbon sourcesmay be combined in the medium.

Satisfactory nitrogen sources include myriad proteinaceous materialssuch as various forms of hydrolysates of casein, soybean meal, cornsteep liquor, distilled solubles, yeast hydrolysates, and the like. Thevarious sources of nitrogen, either alone or in combination, are used inamounts ranging from about 0.2-6% by weight of the aqueous medium.

3,6 Bis (S-chloro-Z-piperidinyl)-2,5-piperazinedione can be recoveredfrom the broth by filtering and concentrating the filtrate under vacuumto about A the original volume and then subjecting the concentratedbroths to extractive procedures.

For example, 3,6-bis-(-chloro-2-piperidinyl) ,-2,5-piperazinedione canbe recovered from the broth or a concentrate thereof by extraction witha water immiscible solvent for the product such as butanol orchloroform. When the broth is extracted at pH 7, the free base isobtained. Alternatively, the broth can be evaporated to dryness andextracted with a suitable solvent such as a loweralkanol, for examplemethanol or ethanol.

Purer forms of the 3,6-bis-(5-chloro-2-piperidinyl)-2,5- piperazinedionecan be obtained by repeated recrystallization from hot methanol. Anotherprocedure which can be utilized comprises absorbing the compound onanion exchange resins with polyalkylamine groups attached to astyrenedivinylbenzene polymer lattice. The absorbed antibiotic isreadily eluted from the resin absorbate with water. Evaporation of theeluate to dryness affords 3,6-bis- (5chloro-Z-piperidinyl)-2,5-piperazinedione which can be further purifiedby fractional recrystallization from methanol.

Alternatively, 3,6-bis-(5-chloro-2-piperidinyl)-2,5-piperazinedione canbe purified by absorption on basic alumina or silica gel and then elutedwith ethylacetate or methanol.

The preferred procedure for purifying 3,6-bis-(5-chloro-2-piperidinyl)-2,5-piperazinedione is as follows:

(A) One part of 3,6-bis-(5-chloro-2-piperidinyl)-2,5- piperazinedione isdissolved in water, filtered, and cooled in an ice bath before adjustingthe pH to 7 with aqueous base. The resultant precipitate is collectedand washed first with water and then with methanol and dried at roomtemperature in vacuo to afford the free base dihydrate of approximately95% purity.

(B) One part of the dried material is added to methanol and warmed on asteam bath. While the solution is still warm additional methanolcontaining excess hydrochloride gas is added and the resultingprecipitate is collected. After washing with methanol and ether theprecipitate is dried at room temperature in vacuo.

(C) One part of the dried material is dissolved in 'water and the aboveprocedures (A) and (B) are repeated to afford a product of approximately98% or greater purity.

(D) One part of product thus obtained is warmed in methanol and theprocedure from (B) to (C) is repeated again to afford an analyticalsample corresponding to the formula C H N Cl O2HCl.

3,6 Bis (5-chloro-2-piperidinyl)-2,5-piperazinedione is a basicsubstance forming acid salts. Thus, the free base,

which can be extracted from the broth at pH 7 on reactions withinorganic or organic acids forms the corresponding acid salt such as thehydrochloride, sulfate, acetate, propionate, and the like.

It contains the elements carbon, hydrogen, nitrogen, oxygen andchlorine. A typical analysis of the hydrochloride salt showed it tocontain 40.06% carbon, 5.79% hydrogen, 13.27% nitrogen, 8.50% oxygen,and 33.39% chlorine. This analysis indicated the molecular formula to beC H N O Cl 2HCl. The hydrochloride salt does not melt below 330 C.

When 3, 6-bis- 5-chloro-2-piperidinyl) -2,5-piperazinedione is reactedwith acetic anhydride in the presence of pyridine at temperatures from 0C. to room temperature, an acetylated derivative or acetate is obtainedmelting at 228 -229 C. with decomposition.

3,6 Bis (5-chloro-2-piperidinyl)-2,5-piperazinedione is soluble inwater, lower alkanols such as methanol, ethanol, and butanol, andchloroform. The free base has low solubility in water at pH 7 butdissolves when heated. The product is soluble in aqueous acid solutionsat pH 2, forming the acid salt.

The free base may be converted to a hydrochloride salt by acidifying amethanolic solution containing the free base with a lower alkanoic(preferably methanolic) solution of hydrogen chloride.3,6-Bis-(5-chloro-2-piperidinyl)-2,5-piperazinedione is stable at roomtemperature for 24 hours in aqueous solution at pH 2 and 10. It islabile after 3-5 minutes at 100 C. at pH 7 aqueous solution. It has beenfound that at 5060 C. the degradation proceeds slowly, some free basestill being detectable after three hours. Acid hydrolysis (6N HCl 16hours at 100 C.) leads to complete degradation.

The novel substituted 3,6-bis-(2-piperidinyl)-2,5-piperazinedionecompounds of this invention can be prepared by several methods. Inaccordance with one embodiment of this invention, novel compounds areprepared by substituting various substituents for the hydrogen atomattached to the nitrogen of the piperidinyl moieties, namely R and R instructure I. Alkylation of the piperidinyl nitrogen can be accomplishedby reacting an alkyl halide with the piperidinyl diketopiperazine. Amongthe alkylating reagents that can be employed in preparing thesecompounds are alkyl halides, for example methyl bromide, ethyl iodide,and propyl iodide; aralkyl halides, for example benzyl chloride,p-bromobenzylchloride, and p-nitrobenzylchloride; and acyloxyalkylhalides, for example acetoxymethyl chloride, propionoxymethyl chloride,pivaloyloxymethyl chloride, and the like. When an alkyl halide or anaralkyl halide is employed as the alkylating reagent, the reaction isrun in an organic solvent, for example acetone, dimethoxyethane,dimethylformamide, and the like. When an acyloxyalkyl halide is employedas the alkylation agent, the reaction is conducted in an organic solventsuch as dimethylsulfoxide, dimethylformamide, dimethoxyethane, andacetone, in the presence of a tertiary amine, for example triethylamine.The temperature for the alkylation reaction is from 0 to 50 C. dependingon the nature of the alkylating agent. The alkylation can be carried outby reacting two molar equivalents of alkylating agent per mole ofpiperidinyl diketopiperazine, however, it is preferable to employ from50 to 100% molar excess of alkylating agent.

The piperidinyl alkylated products may be recovered by methods known tothose skilled in this art, for example, removal of solvent and purifiedby crystallization or chromatography.

Acyl derivatives of the piperidinyl nitrogen of the novel compounds ofthis invention can be prepared by reacting be employed in preparing thenovel acyl compounds include loweralkanoyl halides, for example acetylchloride, propionyl chloride, butyryl chloride, and the like;haloloweralkanoyl halides, for example chloroacetyl chloride, andchloropropionyl chloride; lowercycloalkanoyl halides, for examplecyclopropane carboxylic acid chloride and cyclohexane carboxylic acidchloride; aroyl halides, for example benzoyl chloride, p-chlorobenzoylchloride, pacetamidobenzoyl chloride; alkoxy carbonyl halides, forexample, ethoxycarbonyl chloride, t-butyloxycarbonyl chloride;haloalkyloxy carbonyl halides, for example 3,6,5 trichloroethoxycarbonyl chloride; aralkyloxy carbonyl halide, for examplebenzyloxycarbonyl chloride, pnitrobenzyloxycarbonyl chloride, and thelike. The reaction is conveniently brought about at temperatures of frombetween about C. and 50 C., and preferably from between about 0 C. and20 C. Generally, two moles of acid halide is employed per mole ofpiperidinyl diketopiperazine starting material, however, it ispreferable to employ a 10-50% molar excess of acid halide for the bestresults.

Acylation of the piperidinyl nitrogen can also be accomplished byemploying acid anhydrides, for example acetic anhydride, propionicanhydride, trifiuoroacetic anhydride, glutaric anhydride. The acylationis run in an excess of the anhvdride reagent, for example, from to molesof anhydride per mole of3,6-bis-(5-chloro-2-piperidinyl)-2,5-piperazinedione. The reaction willtake place at a temperature of from 0 to the boiling point of thesolvent. Isolation and purification of the acylated compounds isaccomplished by methods known in the art.

The N-piperidinyl aryl thio derivatives of 3,6-bir-(2-piperidinyl)-2,5-piperazinedione can be prepared by reacting phenylsulfenyl halides with the diketopiperazine starting material.Phenylsulfenyl halides that can be em ployed in the process of preparingthe novel derivatives include phenylsulfenyl chloride, o-nitrophenylsulfenyl chloride, and 2,4-dinitrophenylsulfenyl chloride. The reactionis conducted in organic solvent, for example dimethyl formamide in thepresence of a tertiary amine, for example triethylamine, at atemperature of from 0 to 50 C. The reaction is run with two moles ormore of phenylsulfenyl compound to one mole of piperazine. The productis recovered by removing the solvent followed by solvent extraction withacetone. Purification is accomplished by chromatography.

The nitroso piperidinyl compounds of3,6-bis-(2-piperidinyl)-2,5-piperazinedione are prepared by reactingpiperidinyl diketopiperazine with nitrous acid. The reaction can be runby adding sodium nitrite to an aqueous solution of the piperidinyldiketopiperazine with nitrous acid. The reaction can be run by addingsodium nitrite to an aqueous solution of the piperidinyldiketopiperazine dihydrochloride at ice bath temperatures. The reactionis allowed to stir for 24 hours at room temperature. The N-nitrosocompound is recovered by filtration and purified by recrystallization.

Hydroxyloweralkyl compounds of3,6-bis-(2-piperidinyl)-2,5-piperazinedione can also be prepared bycondensing an alkylene oxide, for example ethylene oxide, propyleneoxide, and the like, with the piperidinyl diketopiperazine. The reactionis run in an alcoholic solvent, for example methanol, by mixing thereactants at a low temperature, for example ice-bath temperature, andthen the reaction is allowed to proceed at room temperature. An excessof alkylene oxide, from 1 to 20 parts per part of piperidinyldiketopiperazine, is employed to prepare the desired compounds. Thedesired product is recovered by filtration and purified byrecrystallization.

Haloloweralkyl compounds of 3,6-bis-(2-piperidinyl)- 2,5-piperazinedionecan also be prepared from the corresponding hydroxyloweralkyl derivativeby reaction with chloromethylenedimethylammonium chloride. The reagentsare combined in dimethylformamide at a low temperature and then allowedto reaction at room temperature for several hours. The crude product isisolated from the reaction mixture by removal of the solvent. The

residual solid is dissolved in water and the product precipidroxide, andpurified by chromatography.

Reaction of a loweralkylsulfonyl halide, for example methanesulfonylchloride, ethanesulfonyl chloride, and the like or an arylsulfonylhalide, for example benzenesulfonyl chloride, p-toluenesulfon'ylchloride and the like, with an hydroxy-piperidinyl-piperazinedione inwhich the piperidinyl nitrogens are substituted, for example 3,6-bis-[1-(o-rritrophenylsulfenyl) 5 hydroxypiperidinyl]-2,5- piperazinedioneaffords the corresponding S-substituted sulfonyloxy compound. One molarequivalent of piperi dinly diketopiperazine is reacted with two molarequivalents, and preferably from 25 to molar excess of sulfonyl halidein pyridine. The sulfonyl halide is added at a low temperature, forexample ice-bath temperature, and the reaction is allowed to proceed tocompletion at room temperature. The sulfonyloxy product is recoveredfrom the reaction mixture by removal of the solvent and purified byrecrystallization from methanol.

Reaction of 3,6-bis-(5-ehloro-2-piperidinyl)-2,5-piperazinedione withsodium hydrogen sulfide affords the corresponding 5 mercapto-piperidinylcompound. Formation of the mercapto compound can be accomplished byreacting one molar equivalent of the starting piperidinyldiketopiperazine compound with two molar equivalents, and preferably anexcess of from 100 to 500% by Weight of sodium hydrogen sulfide. Thereaction is run in an alcoholic solvent, for example ethanol, at atemperature of from 20 C. to the boiling point of the solvent. Thedesired S-mercapto substituted piperidinyl diketopiperazine product isreadily recovered from the reaction mixture by removal of the solventand purified by chromatography.

The chlorine substituent of3,6-bis-'(5-chloro-2-piperidinyl)-2,5-piperazinedione can also bereplaced by a bromine atom by treatment with sodium bromide indimethylsulfoxide. One mole of piperidinyl diketopiperazine is reactedwith an excess, for example 10-50% molar excess, of sodium bromide at 35C. Removal of the solvent and purification by chromatography affords3,6- bis (S-bromo-Z-piperidinyl)-2,5-piperazinedione which can beisolated as the dihydrochloride on treatment with methanol containinghydrogen chloride.

The novel dehydropiperidinyl compounds of this invention are prepared byreacting 3,6-bis-[l-trifluoroacetyl-5-(p-toluenesulfonyloxy)-2-piperidinyl]-1,4-bistrirfluoroacetyl-2,5-piper'azinedione with potassium tertiary butoxidein dimethylsulfoxide. Approximately '10 moles of alkoxide is employedper mole of piperidinyl diketopiperazine starting material and thereaction is heated at 70 C. for several hours. After the solvent isremoved, water is added to the residue. The product which consists of amixture of 3,6-bis-(A'4 and AS-Z-dehydropiperidinyD-2,5-piperazinediones is recovered by filtration and purified bychromatography on silica gel.

Epoxidation of the mixture of dehydropiperidinyl compounds of thisinvention affords a mixture of 3,6-bis(4,5 and5,6-ep-oxy-2-piperidinyl)-2,5-piperazinediones. Reaction of the startingdehydropiperidinyl compound with a perorganic acid such as peracetic,perbenzoic or m-chloroperbenzoic acid, is conveniently brought about attemperatures of between about 0 C. and 30 C. This peroxidation ispreferably carried out in an inert hydrocarbon solvent such aschloroform, methylene dichloride, benzene, toluene, and the like. Thereaction time is not critical, and it is preferable to continue theperoxidation until the maximum amount of epoxide has been produced.Generally, an excess of organic peracid is utilized for best results.The desired products may be recovered by methods known to those skilledin this art, for instance by decomposition of excess perorganic acid andremoval of the solvent.

The 3,6-{4-(3,7-di-aza [4,1,0] heptyl)} and 3,6-{3"(2, 7-diaza [4,1,0]heptyl)}-2,5-piperazinedione compounds of this invention areconveniently prepared by the reaction of iodine isocyanate, preparedfrom silver cyanate and iodine, with a dehydropiperidinyldiketopiperazine compound. The intermediate iodo-isocyanate additioncompound is refluxed with methanol to form the iodocarbmethoxyaminointermediate, which is refluxed with base, for example aqueous potassiumhydroxide to produce the aziridinyl compound. The formation of iodineisocyanate and its reaction with the dehydropiperidinyl diketopiperazineis run in dimethoxyethane. The reagents are combined at a lowtemperature, for example ice-bath temperature, and then the reaction isallowed to run to completion at room temperature. The reaction productis filtered and washed with methanol. The filtrate and washings areconcentrated in vacuo and the residue refluxed with methanol. Afteraddition of aqueous base, the mixture is refluxed for several hours. Thedesired product is recovered by removal of the solvent and purified bychromatography on Florisil.

:Pursuant to another embodiment of this invention, novel compounds of3,6-bz's-(2-piperidin'yl)-2,5 piperazinedione are prepared by replacingvarious groups for the hydrogen atom attached to the nitrogen of thepiperazinedione moiety, that is, R, and R in structure I. In order toprepare the compounds wherein R and R are other than hydrogen, it isfirst necessary to block or protect the piperidinyl nitrogensubstituents at R and R The piperidinyl nitrogens can be blocked byalkylating or acylating with any of the reagents mentioned above forpreparing compounds having substituents at R and .R Alkylation of acompound wherein R and R is an acyl radical can be carried out indimethylformamide with an alkylhalide, for example methyl iodide, in thepresence of sodium hydride. Under these reaction conditions, alkylationof the piperazinedione nitrogens is followed by removal of the ac'ylblocking groups on the piperidinyl nitrogen atoms during work up. Anexcess of reagents, for example three moles of alkylating agent and twomoles of sodium hydride, are employed per mole of diketopiperazine.After the reaction is completed, the solvent is removed and the residueis tritur-ated with dilute base, such as sodium hydroxide. The1,4-alkylated- 3,6-bis-(Z-piperidinyl)-2,5-piperazinedione is isolatedand purified by chromatography.

The novel 3,6-bis-(2-piperidinyl)-2,5-piperazinedione compounds whereinR R R and R are acyl radicals can be prepared by reacting a large excessof an acyl anhydride, for example ten moles per mole of piperazinedionestarting material. The reaction is run in the presence of an acid, forexample the acid from which the anhydride is derived, by refluxing forseveral hours. The 3,6- bis (1 acylated 2 piperidinyl) 1,4 bis acylated-2,5-piperaziuedione compounds are isolated by removing any volatiles andtriturating with an alcohol, for example methanol.

The novel compounds of this invention wherein R and R represent adialkylaminomethyl radical can be prepared by reacting a secondaryamine, for example diethylamine, morpholine and the like, andformaldehyq'e with a piperidinyl diketopiperazine in WhlCh thepiperidinyl nitrogens are substituted with a protecting group such as aloweralkyl radical. The reaction is conveniently run in an alcoholicsolvent, such as ethanol, at reflux.

Although two molar equivalents of formaldehyde and a secondary amine permole of diketopiperazine may be utilized in the process, it ispreferable to run the reaction with from 5-50% molar excess offormaldehyde and amine. The novel compounds are recovered by procedureswell known in the art, for example filtration and purification which canbe accomplished by crystallization or chromatography.

Preparation of compounds wherein R and R are nitroso can be accomplishedby reacting a piperidinyl diketopiperazine which has the piperidinylnitrogens substituted with a protecting group such as a loweralkylradical or acyl radical, with a nitrosylating agent such as nitrosylsulfuric acid. The reaction is conveniently run by adding thenitrosylating agent to the piperidinyl diketopiperazine, for example3,6-bis-(l-methyl-S-chloro 2 piperidinyl)- or3,6-bis-(1-trifluoroacetyl-5-chloro-2 piperidinyl) 2,5- piperazinedionein a mixture of acetic acid and acetic anhydride containing excesssodium metate or potassium acetate. Although the reaction can be carriedout by reacting two molar equivalents of nitrosylating reagent per molarequivalent of piperidinyl diketopiperazine, it is preferable to employfrom 10 to 50% molar excess of nitrosylating agent. The nitrosylatingagent is added at a temperature of from 0 C. to 20 C. and the reactionis allowed to stir for 15 hours at 20 C. The substituted 1,4- dinitrosopiperidinyl diketopiperazine is isolated by removing the volatilecomponents in vacuo and suspending the residue in water and adjustingthe pH to 7 with aqueous base, for example sodium hydroxide.Purification is accomplished by chromatography on silica gel.

When 1,4 dinitroso-3,6-bis-( l-trifluoroacetyl-S-chloro-2-piperidinyl)-2,5-piperazinedione is treated with dilute base, thetrifiuoroacetyl groups can be removed to aflord 1,4 dinitroso 3,6 bis (5chloro 2 piperidinyl)- 2,5-piperazinedione. The reaction is conducted byadding the piperidinyl diketopiperazine to a two phase system ofchloroform and aqueous dilute base, for example 2N sodium hydroxide. Theaddition and reaction are carried out at 0 C. The product is isolated byremoving the solvents in vacuo and purification is by chromatography onsilica gel.

Dehydrohalogenation of3,6-bis-(5-chloro-2-piperidinyl)-2,5-piperazinedione with a strongorganic base, for example 1,5-diazobicyclo-[5,4,0] undec 5 ene, aflords3,6-bis-{2-[1-azabicyclo (3,1,0)-hexane] }-2,5-piperazine-- dione. Thereaction is run by heating the reagents at C. with stirring for 10minutes and then heating on a steam bath for 20 minutes. The mixture isboiled with methanol and after cooling, ether is added to the reactionmixture. After standing several hours, the dehydrohalogenationprecipitates out and the product is isolated by filtration and purifiedby crystallization from methanol.

The preparation of the novel compounds of this invention wherein thepiperidinyl and diketopiperazine nitrogen atoms are joined through amethylene bridging group can be prepared by reacting the3,6-bis-(2-piperidinyl)-2,5-piperazinedione starting compound withformaldehyde in formic acid. The reaction is carried out by dissolvingthe piperidinyl diketopiperazine compound in formic acid and addingformaldehyde with stirring. The reaction is allowed to stand for onehour and the desired product, 3,11-dich1oro-8,16-dioxo (2,3c:5,6C')bis-octa hydro[imidazo (1,5a)pyridino] hexahydropyrazine, isolated byfiltration.

The novel compounds of this invention wherein the 3,6 ppsitions of thediketopiperazine are joined by an equidithio (fiS--S) linkage areprepared by first reacting a completely protected piperidinyldiketopiperazine, for example1,4-dimethyl-3,6-bis-(1-triflu0roacetyl-5-chloro-2-piperidinyl)-2,5-piperazinedione, with phosphorous pentabromide in anorganic solvent, such as o-dichlorobenzene The reaction is run in anexcess of phosphorus pentabromide, for example 3 moles to 1 mole ofdiketopiperazine,

at 150 C. After cooling the reaction, petroleum ether is added and theintermediate bromo compounds precipitates and is collected. Thisintermediate is reacted with a molar equivalent of sodium tetrasulfideby refluxing for hours in an alcohol, such as anhydrous ethanol. Theproduct can be isolated from the reaction mixture by filtering to removeany solids and concentrating the solution in vacuo. The remainingresidue is purified by chromatography on silica gel. Treatment of thepurified material with base, for example dilute ammonium hydroxide,aflords the 3,6- epidithia 3,6 bis (5 chloro 2 piperidinyl) 1,4dimethyl-2,5-piperazinedione. The dihydrochloride can be prepared bytreating the the material purified by chromatography on silica gel withmethanol containing excess hydrogen chloride.

The compounds of this invention wherein R and R are mercapto (SH) can beprepared from the corresponding 3,6-epidithia piperidinyldiketopiperazine, for example,3,6-epidithia-3,6-bis-(5-chloro-2-piperidinyl)-1,4-dimethyl-2,5-piperazinedione by reaction with sodium borohydride. Thereaction is conveniently brought about by adding two moles of sodiumborohydride to one mole of the epidithia compound in an alcoholicsolvent, for example methanol. The addition is made at a lowtemperature, ice-bath temperature, and the reaction mixture is refluxedfor 1 hour. After the solvent is removed, the residue is treated withaqueous base, for example dilute ammonium hydroxide. The dimercaptocom-pound is isolated by methods known in the art, e.g. filtration andfurther purified by chromatography on silica gel. The dihydrochloridecan be prepared by stirring the free base in methanol containing excesshydrogen chloride.

Also included within the scope of this invention are the acid additionsalts of the compounds of this invention formed from such acids ashydrochloric acid, hydrobromic acid, phosphoric acid, acetic acid,ascorbic acid, tartaric acid, maleic acid, and the like.

The hydrochloride salts of the novel compounds of this invention can beprepared by acidifying a methanolic solution containing the free base ofthe compound with hydrogen chloride.

The following examples illustrate methods of preparing the novelcompounds of the present invention. It is to be understood, however,that they are given for the purposes of illustration and not oflimitation.

EXAMPLE 1 3,6-Bis-(5-hydroxy-2-piperidinyl)-2,5-Piperazinedione 1.0 G.of 3,6-bis-(5-chloro-2-piperidinyl)-2,5-piperazinedione is heated in 50ml. of water for /2 hour on a steam bath. The solution is filtered,neutralized with dilute sodium hydroxide and freeze-dried to a whitesolid weighing 1.16 g. Purification of 300 mg. is accomplished bychromatography on 6 x 1000p on silica gel plate using 5:1:2n-butanol:acetic acidzwater as an eluant to give 165 mg. which isdissolved in ml. of hot methanol. Filter solution and bubble in hydrogenchloride gas. Collect precipitate and wash 2X 5 ml. With methanol andml. of ether to give 80 mg. of 3,6bis-(5-hydroxy-2-piperidinyl)-2,5-piperazinedione dihydrochloride.

EXAMPLE 2 3,6-Bis- 1- o-nitrophenylsulfenyl -5-chloro-2-piperidinyl]-2,5-Piperazinedione A 152 mg. portion of3,6-bis-(5-chloro-2-piperidinyl)- 2,5-piperazinedione is stirred in 3ml. of dry dimethylformamide and 165 mg. of o-nitrosulfenylchloride and0.12 ml. of triethylamine are added. After stirring 1 hour, 5 ml. ofchloroform is added. After 2 hours more stirring, the chloroform isremoved in vacuo and the residue filtered before addition of 3 ml. eachof chloroform and dimethylformamide. After addition of 0.070 ml. oftriethylamine and mg. of o-nitrophenylsulfenyl chloride, the mixture isstirred for 15 hours. The solvents are then removed in vacuo and thesolid residue is extracted with 15 ml. of acetone. The acetone extractis concentrated to a solid and extracted with acetone. This process iscontinued until material is obtained which is completely soluble in 15ml. of acetone. The acetone-soluble material is then chromatographed onsilica gel plates. The main band yields 180 mg. of product, m.p. about170-180 C. Recrystallization from methanol yields an analytical sample,m.p. 177179 C., as a methanol sol vate.

When 3,6-bis- 5 -hydroxy-2-piperidinyl) -2,5-piperazinedione is employedin place of 3,6-bis-(5-chloro-2-piperidinyl)-2,5-piperazinedione, thereis obtained 3,6-bis-[1- (o-nitrophenylsulfenyl) S-hydroxy2-piperidinyl]-2,5- piperazinedione.

EXAMPLE 3 3 6-Bis-[ 1- o-nitrophenylsulfenyl)-5-methylsulfonyloxy-2-piperidinyl]-2,5-piperazinedione A pyridinesolution of one mole of 3,6-bzs-[1-(o-nitrophenylsulfenyl)-5-hydroxy2-piperidinyl]-2,5-piperazinedione is cooled and 2.5 moles ofmethanesulfonyl chloride is added. After standing 24 hours, the pyridineis removed in vacuo and the residue is washed with water beforerecrystallization from methanol.

EXAMPLE 4 3,6-Bis- 5-methylsulfonyloxy-Z-piperidinyl] -2,5-piperazinedione Dihydrochloride To a mixture of3,6-bis-[1-(o-nitrophenylsulfenyl)-5- methylsulfonyloxy-Zpiperidinyl]-2,5-piperazinedione in 50-fold methanol is added methanolcontaining excess hydrogen chloride gas. After stirring 12 hours, themixture is warmed briefly. After cooling, the precipitate is collectedand purified by chromatography.

EXAMPLE 5 3 ,6 -B is-( l-chloroac etyl-5-chloro-2-piperidinyl)2,5-Piperazinedione A mixture of 1 mole of3,6-bis-(5-chloro-2-piperidinyl)- 2,5-piperazinedione and parts of drypyridine is stirred and 3.0 moles of chloroacetylchloride is slowlyadded with ice-bath cooling. After stirring one day at room temperature,the mixture is warmed briefly on a steam bath and the solvent is removedin vacuo. Crystallization of the residue from methanol gives3,6-bis-(lchloroacetyl-S-chloro- 2-piperidinyl) -2,5-piperazinedione.

In an analogous manner beginning with 3-chloropropionyl chloride,butyryl chloride, cyclopropanecarboxylic acid chloride,cyclohexanecarboxylic acid chloride, nicotinoyl chloride-hydrochloride,p-chlorobenzoyl chloride, and phthaloylglycyl chloride in place ofchloroacetyl chloride, the corresponding acylated piperidinyl compoundsare prepared 3,6-bis-[1-(3-chloropropionyl)-5-chloro-2-piperidinyl]-2,5-piperazinedione,

3,6-bis- 1-butyryl-5-chloro-2-piperidinyl)-2,5-piperazinedione,

3,6-bis- (cyclopropylcarbonyl-5-chloro-2-piperidinyl) 2,5-piperazinedione,

3,6-bis-( 1-cyclohexylcarbonyl-5-chloro-2-piperidinyl) 2,5-piperazinedione,

3,6-bis-(1-nicotinoyl-5-chloro-2-piperidinyl)-2,5-

piperazinedione,

3,6-bis-( 1-p-chlorobenzoyl-S-chloro-2-piperidinyl) 2,5-piperazinedione,and

3,6-bis-( 1-phthaloylglycyl-5-chloro-2-piperidinyl) 2,5-piperazinedione.

1 1 EXAMPLE 6 3 ,6-Bis- 1-glycyl-5-chloro-2-piperidinyl2,5-Piperazinedione One mole of 2,6-bis-(l-phthaloylglycyl 5 chloro-2-piperidinyl)-2,5-piperazinedione and 5-6 moles of hydrazine hydrate arerefluxed in 200 parts of ethanol for 5 hours. The solvent is removed invacuo and the residue is washed with small amounts of water before beingpurified by chromatography on silica gel.

EXAMPLE 7 3 ,6-[1-tetrahydro-2H-1,3,2-oxazaphosphorin-2-yl)-5-chloro-2-piperidinyl] 2,5 Piperazinedione A mixture of one mole of3,6-bis(5-chloro-2-piperidinyl)-2,5-piperazinedione and 1.5 moles oftriethylamine is stirred in 200 parts of acetone and 2.2 moles of 2chloro-tetrahydro-ZH-1,3,2-oxazaphosphorine p-oxide is added slowly inparts of methylene chloride. After stirring 5 hours at room temperatureand 24 hours at reflux, the solvents are removed in vacuo and theresidue is washed with small amounts of dilute hydrochloric acidfollowed by water. The residue is then purified by chromatography onsilica gel to afford 3,6-[1-(tetrahydro2H-l,3,2-oxazaphosphorin-2-yl)-5-chloro-2 piperidinyl]-2,5-piperazinedione.

EXAMPLE 8 3,6-Bis-(5-mercapto-2-piperidinyl)-2,5-Piperazinedione Onemole of 3,6-bis(5-chloro-2-piperidinyl)-2,5-piperazinedione is added to2.2 moles of sodium hydrogen sulfide in 50 parts of ethanol. The mixtureis stirred one day at room temperature and then refluxed hours. Thesolvent is removed in vacuo and the residue purified by chromatographyon silica gel to afford 3,6-bis-(5-mercapto-2-piperidinyl)-2,5-piperazinedione.

EXAMPLE 9 3,6Bis-(1-methyl-5-chloro-2-piperidinyl)-2,5- PiperazinedioneA 1.0 g. portion of 3,6-bis(5-chloro-2-piperidinyl)-2,5- iperazinedioneis stirred in 150 ml. of acetone and -8 ml. of methyl bromide for 4days. The mixture is concentrated in vacuo to a solid which is stirredfor an additional 5 days in 125 ml. of acetone and 10 ml. of methylbromide. After concentration in vacuo the residue is dissolved in 25 ml.of dilute hydrochloric acid and filtered. The pH is then adjusted to 7.1with 1N sodium hydroxide and .88 g. of solid is collected; extractionwith 3 x 10 ml. of 10% methanol/chloroform gives .35 g. of solidmaterial. Chromatography on 4 X 1000 silica gel plates (10%methanol/chloroform) gives 175 mg. of product which is slurried in ml.of 1:5 methanokchloroform and is heated with gaseous hydrogen chloride.After adding 5 ml. of ether, 141 mg. of the dihydrochloride of 3,6-bis-(l-methyl-5-chloro-2-piperidinyl)-2,5 piperazinedione is collected.

EXAMPLE 10 3 ,6-Bis- 1-benzyl-5-chloro-2-piperidinyl -2, 5-

Piperazinedione One mole of3,6-bis(5-chloro-2-piperidinyl)-2,5-plperazinedione is stirred in 150parts of acetone and 2.2 moles of benzyl bromide is added. The mixtureis stirred 24 hours and refluxed 24 hours. The solvent is removed invacuo and treated as in the preparation of3,6-bis-(lmethyl-S-chloro-Z-piperidinyl -2,5-piperazinedione.

l 2 EXAMPLE 11 3,6-Bis-(S-bromo-Z-piperidinyl) -2,5-PiperazinedioneDihydrochloride EXAMPLE 12 3 ,6-Bis-(1-pivaloyloxymethyl-5-chloro-2-piperidinyl 2,5-Piperazinedione One moleof 3,6-bis(5-chloro-2-piperidinyl)-2,5-piperazinedione is stirred in 10parts of dry dimethylsulfoxide with 3 moles of triethylamine and 3 molesof pivaloyloxymethyl chlorides is added. After stirring one week thesolvent is removed in vacuo and purification is achieved bychromatography on silica gel.

EXAMPLE l3 1,2-Bis- N-morpholinomethyl -3 ,6-Bis-( l-methyl-S-chloro-2-piperidinyl -2,5-Piperazinedione One mole of3,6-bis-(1-methyl-5-chloro-2-piperidinyl)- 2,5-piperazinedione, 2.1moles of formalin and 2.1 moles of morpholine are refluxed in 5 parts ofethanol for 15 minutes. After cooling the product is collected andpurified by recrystallization from methanol.

EXAMPLE 14 3,6-Bis-[ l-trifluoroacetyl-S-(p-toluenesulfonyloxy)-2-Piperidinyl] -l ,4-Bis-Trifluoroacetyl-2,5-

Piperazinedione One mole of3,6-bis-[5-(p-toluenesulfonyloxy-Z-piperidinyl]-2,5-piperazinedioneprepared from 3,6-bis-[1-(onitrophenylsulfenyl) 5 hydroxy 2 piperidinyl]2,5- piperazinedione according to the procedure of Example 4 is refluxedwith 10 moles of trifluoroacetic anhydride and 5 moles oftrifluoroacetic acid for 15 hours. The excess solvent and reagent areremoved in vacuo and the residue trituratecl with methanol to afford thedesired product.

EXAMPLE 15 Mixture of 3,6-Bis-[A4 and AS-Z-dehydropiperidinyl)-2,5-

Piperazinediones One mole of 3,6-bis-[l-trifluoroacetyl-S(p-toluenesulfonyloxy)-2-piperidinyl]-1,4-bis trifluoroacetyl 2,5-piperazinedione is dissolved in 20 parts of dry (distilled from bariumoxide) dimethylsulfoxide and 10 moles of freshly prepared potassiumtertiary butoxide is added. The mixture is stirred at for 2 hours, andthe solvent is removed in vacuo. Water is cautiously added to theresidue and after stirring one hour at room temperature, the solid iscollected and the olefin fraction purified by chromatography on silicagel.

EXAMPLE 16 Mixture of 3,6-Bis-(A4 and A5-2-dehydro-1-trifluoroacetyl 2piperidinyl) 1,4-Bis-Trifluoroacety1-2,S- Piperazinediones One mole of3,6-bz's- [A4 and A5-2-dehydropiperidinyl)- 2,5-piperazinedione isrefluxed 10 hours with 10 moles of trifluoroacetic anhydride and 2 molesof trifluoroacetic acid. The volatiles are removed in vacuo and theresidue triturated with methanol to afiord a mixture of 3,6-bis- 13 (A4and AS-Z-dehydro-l-trifiuoroacetyl-2-piperidinyl)-1,4-bis-trifluoroacetyl-2,5-piperazinediones.

EXAMPLE 17 Mixture of 3,6-bis- (4,5- and 5,6-epoxy-2-piperidinyl)-2,5-Piperazinediones One mole of 3,6-bis-(A4 andAS-Z-dehydro-l-trifluoroacetyl 2piperidinyl)-1,4-bis-trifiuoroacetyl-2,5-piperazinediones is stirred in200 parts of chloroform with 5.0 moles of m-chloroperbenzoic acid fortwo weeks. The chloroform is removed in vacuo and the residue is stirredwith excess dilute base and the insolubles are collected. Purificationby chromatography on Florosil produces a mixture of 3,6-bis-(4,5- and5,6-epoxy-2-piperidinyl)-2,5-piperazinedione.

EXAMPLE 18 Mixture of 3,6-[4-(3,7 diaza [4,1,0]heptyl and 3,6-[3-2,7-diaza [4,1,0]heptyl] 2,5-Piperazinedione To a stirred mixture of 2.2mmoles of iodine and 3.0 mmoles of silver cyanate are stirred in 10 ml.of dry dimethoxyethane with ice-bath cooling and 1.0 mole of a mixtureof 3,6-bis-(A4 and A5-2-dehydropiperidinyl)- 2,5-piperazinedione isadded. .After stirring 1 hour at and 1 week at room temperature, themixture is filtered through celite. The collected solid is then washedwell with dry methanoL'The filtrate and washings are concentrated invacuo and the residue stirred and refluxed for 12 hours in 25 ml. of drymethanol. 2.0 Mmoles of 10% potassium hydroxide is added and the mixtureis refluxed 3 hours more. The solvent is then removed in vacuo and theresidue is washed with water and purified by chromatography on Florosil.

EXAMPLE l9 3,6-Bis-( 1-trifluoroacetyl-S-chloro-Z-piperidinyl) -1,4-Bis-Trifiuoroacetyl-Z, -Pip erazine dione One mole of 3,6 bis (5 chloro2 piperidinyl)-2,5- piperazinedione is stirred 12 hours with moles oftrifluoroacetic anhydride and 5 moles of trifluoroacetic acid. Themixture is then refluxed 15 hours and the volatiles are removed invacuo. The residue is triturated with methanol.

EXAMPLE 3,11-Dichloro-8,16-Dioxo(2,3C:5,6,C')Bis-{Octahydro- [imidazo1,5 a) Pyridino] }Hexahydropyrazine One mole of 3,6 bis (5 chloro 2piperidinyl)-2,5- iperazinedione is dissolved in one part of 95-1'00%formic acid and 0.7 parts of 37% formaldehyde is added with stirring.After standing one hour, the insoluble material is collected byfiltration and Washed with ether to afiord 3,11 dichloro8,16-dioxo(2,3C:5,6,C') bis-{octahydro [imidazo (1,5a)pyridino}hexahydropyrazine.

EXAMPLE 21 3,6-Bis( l-methyl-1-oxo-5-chloro-2-piperidinyl)2,5-Piperazinedione One mole of3,6-bis-(1-methyl-5-chloro-2-piperidinyl)- 2,5-piperazinedione isstirred in 200 ml. methanol and the mixture is cooled with an ice bathbefore 2 moles of hydrogen peroxide is added dropwise. After stirring 2hours at room temperature, another 2 moles of 30% hydrogen peroxide areadded and after 15 hours stirring, another 2 moles of hydrogen peroxideare added. After stirring 15 hours, excess peroxide is decomposed withplatinum black. The mixture is filtered and concentrated in vacuo to aresidue which is purified by chromatography on Florosil.

- EXAMPLE 22 3,6-Bis(l-benzyl-l-oxo-5-chloro-2-piperidinyl)-2,5-Piperazinedione One mole of3,6-bz's-(l-benzyl-S-chloro-Z-piperidinyl)- 2,5-piperazinedione isstirred in 200 ml. methanol and the mixture is cooled with an ice bathbefore 2 moles of 30% hydrogen peroxide is added dropwise. Afterstirring 2 hours at room temperature, another 2 moles of 30% hydrogenperoxide are added and after 51 hours stirring, another 2 moles ofhydrogen peroxide are added. After stirring 15 hours, excess peroxide isdecomposed with platinum black. The mixture is filtered and concentratedin vacuo to a residue which is purified by chromatography on Florosil.

EXAMPLE 23 3,6-Bis[ 1- (pfiB-trichloroethoxycarbonyl) -5-Chloro-Z-Piperidinyl] -2,5-Piperazinedione One mole of 3,6 bis-(5 chloro2-piperidinyl)-2,5- iperazinedione is stirred in 200 parts of acetonewith 3 moles of triethylamine and 3 moles ofpfip-trichloroethoxycarbonyl chloride in parts of acetone are addeddropwise with stirring over a period of one hour. The mixture is stirredone day and the volatiles are removed in vacuo and the residue washedwith water before purification by chromatography on silica gel.

When propoxycarbonyl chloride and benzyloxycarbonyl chloride areemployed in place of pfifl-trichloroethoxycarbonyl chloride in the aboveprocedure, 3,6-bis- (1 propoxycarbonyl 5 chloro 2 piperidinyl)-2,5-iperazinedione and 3,6 bis (l-benzyloxycarbonyl-S-chloro-2-piperidinyl)-2,5-piperazinedione are obtained.

EXAMPLE 24- 3,6-Bis( 1-trifluoroacetyl-5-chloro-2-piperidinyl) -2,5-Piperazinedione One mole of 3,6 bis (5-chloro-2-piperidinyl)-2,5-piperazinedione is stirred in 100 parts of pyridine and 5 moles oftrifluoroacetic anhydride are added. After stirring 15 hours, thevolatiles are removed in vacuo and the residue is crystallized frommethanol.

EXAMPLE 25 1,4-Dimethyl-3,6-Bis-(5-chloro-2-piperidinyl)-2,5-Piperazinedione One mole of 3,6 bis-(l-trifiuoroacetyl-S-chloro-Z-piperidinyl)-2,5-piperazinedione is dissolved in 50 parts of drydimethylformamide. The solution is stirred under a nitrogen atmosphereand 2 moles of sodium hydride are added. After stirring 5 hours, 3 molesof methyliodide are added. The mixture is stirred 15 hours and thesolvent is removed in vacuo. The residue is triturated with dilutesodium hydroxide, stirred in dilute sodium hydroxide for one hour, andthe solid is collected and purified by chromatography on silica gel toafford l,4-dimethyl-3,6-bis-(5-chloro-Z-piperidinyl)-2,5-piperazinedione.

EXAMPLE 261,4-Dimethyl-3,6-Bisl-trifluoroacetyl-5-chloro-2-piperidinyl)-2,5-PiperazinedioneOne mole of1,4-dimethyl-3,6-bis-(5-chloro-2-piperidinyl)-2,5-piperazinedione isstirred in 100 parts of pyridine and 5 moles of trifluoroaceticanhydride are added. After stirring 15 hours, the volatiles are removedin vacuo and the residue is crystallized from methanol.

EXAMPLE 273,6-Epidithia-3,6-Bis-(5-chloro-2-piperidinyl)-1,4-Dimethyl-2,5-PiperazinedioneDihydrochloride One mole of l,4-dimethyl-3,6-bis-(l-trifiuoroacetyl-S-chloro-2-piperidinyl)-2,5-piperazinedione is stirred in 15 parts ofo-dichlorobenzene with 3 moles of phosphorous 15 pentabromide. Themixture is heated to 150 for 15 minutes, cooled and diluted withpetroleum ether. The precipitate is collected, dried in vacuo andrefluxed hours with an equivalent of sodium tetrasulfide in 20 parts ofanhydrous ethanol. The mixture is then filtered and the filtrateconcentrated in vacuo to a residue which is purified by chromatographyon silica gel. The purified material is then stirred in excess diluteammonium hydroxide and collected on a filter. The dihydrochloride isprepared by stirring in methanol containing excess hydrogen chlorideafter the free base has been purified by chromatography on silica gel.

EXAMPLE 28 3,6-Dimercapto-3 ,6-Bis-(5-chloro-2-piperidinyl) -1,4-Dimethyl-2,5-Piperazinedione Dihydrochloride To one mole of3,6-epidithia-3,6-bis-(5-chloro-2-piperidinyl)-1,4-dimethyl-2,5-piperazinedionein 25 parts of methanol is added portionwise, with stirring, and icebath cooling, 2.0 moles of sodium borohydride. After stirring one hourat room temperature the mixture is refluxed one hour. The solvent isremoved in vacuo and the residue is stirred with 20 parts of water andexcess dilute ammonium hydroxide. The solid is then collected on afilter and purified by chromatography on silica gel. The dihydrochlorideis prepared by stirring in methanol with excess hydrogen chloride.

EXAMPLE 29 3,6-Bis-{2-[ l-azabicyclo (3,1,0)hexane}-2,5-PiperazinedioneA 1.50 g. portion of finely ground 3,6-bis-(5-chloro-2-piperidinyl)-2,5-piperazinedione is mixed with 2.25 ml. of1,5-diazobicyclo[5,4,0]undec-5-ene and heated in an oil bath at 95 C.,with stirring, for minutes. The mixture is then stirred and heated on asteam bath for minutes. A ml. portion of methanol is added and themixture is brought to a boil. After cooling, 10 ml. of ether is addedand the mixture is allowed to stand 15 hours. C01- lect .54 g. Let standand collect .14 g. more. Recrystallize both crops from ethanol to give.47 g. of 3,6-bis- {2- l-azabicyclo (3,1,0) -hexane]}-2,5-piperazinedione.

EXAMPLE 3o3,6-Bis-(N-nitroso-5-chloro-2-piperidinyl)-2,5-Piperazinedione Asolution of 1.5 g. of 3,6-bis-(5-chloro-2-piperidinyl)-2,5-piperazinedione dihydrochloride in 200 ml. of water is cooled in anice bath and 1.0 g. of sodium nitrite is added. The cooling bath isremoved and the mixture is stirred 24 hours. The precipitate iscollected, 804 mg., boiled in 100 ml. of hot methanol, and filtered. Thefiltrate is concentrated to 50 ml. and allowed to cool. .52 G. of3,6-bis- (N nitroso-S-chloro-2-piperidinyl)-2,5-piperazinedione iscollected.

EXAMPLE 31 3 ,6-Bis- S-chloro- 1- (Z-hydroxyethyl) -2-piperidinyl] -2,5-

Piperazinedione A solution of one part 3,6-bis-(5-chloro-2-piperidinyl)-2,5-piperazinedione in 250 parts of methanol is cooled in an ice bathand 10 parts of ethylene oxide are condensed into the solution. Afterstanding 2 weeks the mixture is concentrated in vacuo to a solid whichis warmed in 10 parts of methanol. After cooling 0.5 parts of product iscollected. Further purification is achieved by recrystallization frommethanol or my conversion to the dihydrochloride. The dihydrochloride isprepared by warming in parts of methanol containing excess hydrogenchloride. After cooling, the solid is collected, washed 4 x 10 partswith methanol, and dried under vacuum.

1 6- EXAMPLE 32 3,6-Bis- [S-chloro-l- (Z-chloroethyl -2-piperidinyl]-2,5- Piperazinedione 136 Mg. of freshly preparedchloromethylenedimethylammonium chloride is dissolved in 3 ml. of drydimethylformamide and stirred under a nitrogen atmosphere with ice-bathcooling before 205 mg. of 3,6-bis-[5-chloro-2-(2- hydroxyethyl)-2-piperidinyl] -2,5-piperazinedione is added. The cooling bath isremoved and the mixture is stirred 15 hours. The solvent is removed invacuo and the residual solid is dissolved in 2 /2 ml. of water andfiltered. The filtrate is brought to pH 7 with 1N sodium hydroxide andthe resultant precipitate is collected. Purification is achieved bychromatography on preparative silica gel thin layer plates using 10%methanol/ chloroform as an eluant to give 100 mg. The dihydrochloride isprepared by slurrying one part of the free base in 100 parts of 5:1methanol: chloroform and bubbling in hydrogen chloride. To the resultantsolution is added 20 parts ether and the precipitate is collected afterstanding for one hour; 100 mg. of di hydrochloride with /s mole of etheras a solvent is obtained.

EXAMPLE 33 l,4"Dinitroso-3,6Bisl-methyl-5-chloro-2-piperidinyl)2,5-Piperazinedione A 2 mmole portion of 3,6-bz's(1-methyl-5-chloro-2-piperidinyl)-2,5-piperazinedione is stirred in a mixture of 10 ml. ofacetic acid, 5 ml. of acetic anhydride, and 3 mmole of sodium acetateand 2.5 mmole of nitrosyl sulfuric acid is added portionwise in 5minutes. After stirring 15 hours the acetic and acetic anhydride isremoved in vacuo and the residue is slurried with water and brought topH 7 with cold 1N sodium hydroxide. Purification is achieved bychromatography on silica gel tlc plates.

When 3,6-bis-( 1-trifluoroacetyl-5-chloro-2-piperidinyl)2,5-piperazinedione is employed in place of 3,-6-bis-(1-methyl-5-chloro-2 piperidinyl)-2,5-piperazinedione, there is obtained1,4-dinitroso 3,6 bis-(l-trifluoroacetyl-S-chloro-2-piperidinyl)-2,5-piperazinedione.

EXAMPLE 34 1,4-Dinitroso-3,6-Bis- 5-chloro-2-piperidinyl) -2,5-Piperazinedione Preparation of 3,6-Bis- (5-chloror2-piperidinyl)-2,5-Piperazinedione A lyophilized culture of 3,6-bis-(5-chloro 2piperidinyl) 2,5 piperazinedione (Streptomyces griseoluteus NRRL 3412)is suspended in 2 ml. of a medium having the following composition:

Yeast extract g 10 Glucose g 10 Phosphate buffer* ml 20 MgSO .7H O g 0.5Distilled water ml 1000 *Phosphate buffer I QPOl q 91 NaaHPos 2 95 To1000 ml. with distilled water.

Percent Dextrose 1 N-Z amine 1 NaCl 0.5 Meat extract 0.3

Distilled water, q.s. ad.

and the growth on the slant is scraped into the suspension and used toinoculate a 250 ml. baflled Erlenmeyer flask containing 50 m1. of thesame medium. The inoculated flask is then placed on a rotary shaker andincubated at 28 C. for 72 hours or until good vegetative growth isobtained.

An inoculum of 10 ml. of the resulting vegetative growth is then used toinoculate a 2 L. baffled Erlenmeyer flask containing 500 ml. ofsterilized medium of the same composition as shown above, and theinoculated flask is then placed on a rotary shaker and incubated for72-96 hours at 28 C. or until good vegetative growth is obtained.

The resulting fermentation broth is used to inoculate a 50 gallonstainless steel fermentor containing 160 L. of the medium of the samecomposition shown above. The inoculated medium is incubated at 28 C.with agitation at 150 r.p.m. while maintaining an air flow of 3 c.f.m.through the fermentation broth. During the 72-96 hour fermentationperiod, small amounts of an antifoamant (Polyglycol 2,000) is added tocontrol foaming of the batch.

8.3% of the resulting broth is then used to inoculate a 200 gallonstainless steel fermentor containing 440 L. of a medium having a pH offrom 7 to 7.2 and having the following composition:

Distilled water, q.s. ad.

The inoculum is incubated for 120 to 160 hours with agitation at 130r.p.m. while maintaining an air flow of 10 c.f.m. through the broth, adefoamer being added if necessary.

Filtered broth (100 gal.) obtained by the fermentation proceduredescribed above is filtered and concentrated in vacuo to approximately16 gallons and a gallon portion of the concentrated broth islyophilized.

1 Kg. of lyophilized filtered broth representing a lyophilized portionfrom a 16 gallon concentrated broth obtained above is suspended in 5 L.of absolute ethanol. The mixture is then filtered to remove insolublematerial and the filtrate subsequently concentrated to an aqueoussolution.

The insoluble residue from the initial ethanol extraction is extractedwith 2500 ml. of absolute ethanol. The ethanol soluble fraction afterfiltration is concentrated in vacuo to a small volume. A crystallinefraction is observed salting out and removed by centrifugation,suspended in water and lyophilized.

A small amount of the crystalline fraction obtained is mixed with about5 ml. water and about ml. of methanal and concentrated to dryness afteradding about 10 ml. of methanol two times. The final solids are thendissolved in 2535 ml. of absolute methanol and allowed to stand at roomtemperature for 12 hours after which a small amount of crystallinematerial separates out. The sample is then cooled to 45 C. for four daysand the resulting crystalline fraction is removed by centrifugation anddried in vacuo. The crystalline material thus obtained decomposed at 330C. leaving a brown residue.

Mg. of crystalline sample is dissolved in N/10:hydrochloric acid, thenneutralized slowly with N/10 sodium hydroxide to convert it to the freebase. A precipitate forms at about pH 7. It is collected in a centrifugetube and dried in vacuo to yield 54 mg. of 3,6-bis-(5-chloro-2piperidinyl)-2,5-piperazinedione as the free base. Analysis: Calculatedfor C H N O Cl Cl, 20.3%. Found: Cl, 19.07%.

The free base thus obtained is dissolved in methanol, a trace ofinsoluble impurity is separated and the solution is acidified byaddition of alcoholic hydrogen chloride. The solvent is evaporated atreduced pressure to a small volume. On standing, white crystals of thehydrochlon'de form. The crystals are purified by recrystallization frommethanol and dried in vacuo. No melting poin is observed below 330 C.

Anal. Calcd. for C H N O Cl -2HCl: C, 39.8; H, 5.74; N, 13.25; 0, 7.59;Cl, 33.5. Found: C, 40.23; H, 5.85; N, l2.76;0, 8.5; CI, 32.82.

Assay The novel 3,6 bis (2-piperidinyl)-2,5-piperazinedi0ne compounds ofthis invention may be conveniently assayed for anti-tumor activity usingthe Human Tumor- Egg Host System, tumor implants of human epidermoidcarcinoma (H. Ep. #3) are placed on the chorioallantoic membranes often-day embryonated eggs. The eggs are incubated one day, and thoseshowing positive takes are selected for the test. The3,6-bis-(2-piperidinyl)-2,5-

piperazinedione compound is then injected onto the chorioallantoicmembranes of the egg. Seven days after injection, the eggs are harvestedand tumors and embryos from treated and untreated control groups areweighed and the percent growth inhibition of the tumor and embryo in thetreated egg is obtained as follows:

Ten tumor implanted eggs are sacrificed at the time of injection withthe 3,6-bis-(2-piperidinyl) 2,5-piperazinedione compound to determinemean weight of the tumor. The value obtained is then subtracted from themean weight obtained from the treated and untreated control tumors atthe time of the harvest to determine the actual increase in weight ofthe tumors during the treatment period. The percent growth retardationfor treated eggs is obtained by comparing the increase in weight oftreated tumors with the increase in weight of untreated control tumorsusing the formula (-T/C 2 100). The percent growth retardation forembros is determined in a similar manner.

After determining the effect of treatment on primary growth of the H.Ep. #3 tumor, a portion of lung tissue from each harvested embryo isimplanted into a fresh group of 10-day eggs. The eggs are incubated for8 days and then weighed the resulting tumors to obtain an estimate ofthe amount of metastasis to the lung.

The anti-tumor activity of the 3,6-bi.s .-(2-piperidinyl)-2,5-piperazinedione compounds of this invention employing the assay withhuman epidermoid carcinoma (H. Ep. #3) are found in Table l.

TABLE 1 Biological assay in eggs against H. Ep. #3 tumor Percentreduction Dose, Name mg. [egg Deaths Embryo Tumor Metastasis 3,11-dichloro-8, 16-dioxo-(2,3e:5. 60) his [octahydro [imidazo (1, 5a)py1idin0]} hexahydropyrazine- 5 /6 23 28 104 l. 6 0/6 6 1 95 0.5 0/6 4 939 3, 6-bia-[5-ehloro-1-(Z-hydroxyethyl)2-piperldinyl] 2,-piperazinedi0ne. 1. 25 4/6 37 96 101 0. 42 1/6 10 38 62 0. 14 3/6 -9 1935 3, 6-1;ia-[l-(o-nitrophenylsulienyl)5-chloro-2-pieridinyIl-2,5-piperazinedione 3 0/3 16 26 28 1 0/3 8 5 47 3 0/6 5 29 30 3,6-m-(1-methyl-5-chloro-2-piperldinyl)2, 5-piperzainedi0ne 1 0/4 29 so100 0. 33 0/4 36 100 0. 3 0/6 4 5 56 0. 1 0/6 11 30 28 3,fi-bz's-(Z-[l-azabieyclo (3, 1, 0)l1exane] }-2, 5-piperazinedi0ne 0. 091/6 39 79 102 0. 03 0/ 6 15 33 92 0.01 0/ 6 2 77 3,G-bia-[B-ehloro-l-(2-chloroethyl)2-piperidinylI-2, 5-piperazinedione 10/6 42 86 99 0.33 1/6 84 100 0. 11 1/6 18 32 95 037 0/6 11 40 71 I 01233/6 0 40 53 3, fi-bia (5-hydroxy-2-piperidiny1)2, S-piperazinedione 30/4 7 59 92 1 0/4 16 12 92 0. 33 0/4 19 33 63 1 2/ 6 8 19 97 Theanti-metastasis activity using H. Ep. #3 tumor cells iru ova of the3,6-bis-(2-piperidinyl)-2,5-piperazinedione compounds of this inventionis compared against 3,6-bis-(5-chloro 2 piperidinyl) 2,5-piperazinedioneand other known anti-tumor agents, for example cyclophosphonamide,1,3-bis-(2-ch1oroethyl) 1 nitrosourea (BCNU) and cytosine arabinoside.Table 2 gives the activities relative to 3,6-bis-(5-ch1oro 2piperidinyl) 2,5- piperazinedione as 100 in this anti-metastasis assay.

TABLE 2 Anti-Metastasis Assay in Eggs Against H. Ep. #3 Tumor Cells3,6-bis-(5-chloro 2-piperidinyl) 2,5 piperazinedione 100.Cyclophosphonamide Toxic. BCNU 3. Cytosine arabinoside 20.3,6-bis-(5-hydroxy 2 piperidinyl)-2,5-piperazinedione. 1.3,6-bis-(1-methyl 5 chloro 2 piperidinyD- 2,5-piperazinedione. 10-3,6-bis-[5-ch1oro 1 (2-hydroxyethy1)-2-piper idinyl]2,5-piperazinedione. 2. 3,6-bis-[5-chloro 1(2-ch1oroethyl)-2-piperidinyl] 2,5-piperazinedione. 30.3,6-bis-[l-(o-nitrophenylsulfenyl) 5 chloro- 2-piperidinyl]2,5-piperazinedione. 1. 3,6-bis-(1-nitroso 5 chloro 2 piperidinyl)-2,5-piperazinedione. 2. 3,6 bis {2-[l-azabicyclo(3,1,0)hexane] }-2,5-

piperazinedione. 100-200'. 3,11 dichloro 8,16-dioxo (2,3c:5,6c')bis-{octahydro[imidazo(1,5a)pyridino]} hexahydropyrazine. 2.

As indicated above, the 3,6-bis-(2-piperidinyl)-2,5-piperazinedionecompounds of this invention possess antitumor activity and areconvenient reference compounds for the testing of other compounds forthis activity.

What is claimed is: 1- A compound of the structure wherein R and R areeach hydrogen, lower alkyl of 1 to 6 carbon atoms, lower alkanoyl of 2to 4 carbon atoms, dilower alkylaminomethyl wherein the lower alkylscontain 2 to 5 carbon atoms; R; and R are each hydrogen or mercapto.

2. The compound of the structure designated3,6-bis-{2-[l-azabicyclo(3,1,0)hexane]} 2,5- piperazinedione.

References Cited 260-2475, 250, 268 C, 268 BC; 424248, 250

1. A COMPOUND OF THE STRUCTURE